Ultrapower

DNA extractions were performed using commercial DNA extraction kits (Qiagen^®, QIAamp^® DNA Blood Mini Kit, Maryland, USA), according to the manufacturer’s instructions. PCR was conducted using TBR1/2 primers to amplify a 164 bp product from a highly repeated sequence of mini-chromosome satellite DNA for the detection of Trypanozoon (Table-1), following a protocol previously described [21 (link),22 (link)]. Briefly, 18 μL PCR mixtures were prepared using PCR buffer, 50 mM MgCl₂, 10 mM dNTPs mixed, 10 μM of each primer, 0.5 Unit of taq DNA polymerase (Invitrogen™, Life Technologies, California, USA), and 2 μL of DNA sample. Trypanosoma evansi DNA was extracted from the blood of an experimentally infected mouse for the positive control, and distilled water was used as the negative control. Initial denaturation was 94°C 1 min followed by 30 cycles of denaturation at 94°C 30 s, annealing at 60°C 1 min, extension at 72°C 30 s, and an extra final extension at 72°C 2 min. PCR product was stained using non-toxic nucleic acid stains (Ultrapower^®, BioTeke, Beijing, China) and migrated through 1.5% agarose gel (Biotech™, Bio Basic Canada Inc., Ontario, Canada) in a submarine electrophoresis system (Advance Mupid-exU TM, Takara Bio Inc., California, USA). PCR products were observed under a UV transilluminator (InGenius™, Syngene Bio Imaging^®, Labnet International Inc., Woodbridge, USA).

The 8% poly/acrylamide gel was used for the native electrophoresis analysis and run at 4°C. 1× TBE and 2-(N-morpholino) ethanesulfonic acid monohydrate (MES, pH 4.5, 50 mM) buffer solutions were used to prepare the neutral and acid gels, respectively. The running buffers were further supplemented with 50 mM LiOAc for the neutral gel and 100 mM KOAc for the acid gel. The gel electrophoresis was performed for 3.5 h at 100 V. Results were observed using an Ultrapower™ visible light transilluminator (Bioteke, Beijing, China) after using GelGreen dye (Biotium) in 100 mM NaCl. The gel image was processed with Adobe Photoshop CS6 software to reduce background interference [13 (link)].

Genomic DNA from each alcohol-preserved mosquito was extracted using DNAzol® reagent (Invitrogen, Carlsbad, California, USA). After PCR reactions, the amplified products were analyzed on 3% agarose gel with a low molecular weight DNA ladder (New England Biolab, Ipswich, Massachusetts, USA) used to estimate the band size. The electrophoresis was run for 50 min at 100 V in TBE buffer. The gel was then submerged in 0.5 μg/ml ethidium bromide (EtBr) (Invitrogen) solution for 15 min, de-stained for 5 min in distilled water, and visualized in a UV transilluminator.
Since EtBr is known to be a strong mutagen and is treated as hazardous waste, as alternatives we tried using Ultrapower™ (BioTeke, Beijing, China) and RedSafe™ (iNtRON Biotechnology, Gyeonggi-do, Korea) dyes, which are advertised as non-toxic and have no hazard waste. For Ultrapower™ staining, the dye solution (10,000×) was diluted 100-fold in 6× loading dye (New England Biolab), then 1 μl of diluted dye was mixed with 5 μl of PCR product. 1 μl of diluted dye was also added to 5 μl of the DNA ladder before loading on the gel. For RedSafe™ staining, 5 μl of this dye (20,000×) was mixed in with 100 ml molten agarose gel prior to gel pouring. Visualization was done immediately after gel electrophoresis.